Recent findings in this laboratory indicate that signals which lead to elevation of intracellular Ca++ and activation of protein kinase C can effect the phosphorylation and turnover of the ets proteins, which are found primarily in the nucleus. Thus, our current model for ets function in the cell envisages ets proteins as intermediary signal transducers which respond rapidly to signals generated by second messengers in the cytoplasm and are somehow involved in relaying this information to effect nuclear events. In this research we are attempting to test and clarify this idea and are currently focusing on two major areas: 1) the possible regulation of ets during the cell division cycle, and 2) the role of ets in intracellular signal transduction in the astrocyte lineage. The cell cycle project utilizes centrifugal elutriation to fractionate CEM (human T-cell leukemia) cells into populations staged at different phases of the cell cycle; ets proteins in staged cells are then analyzed. Progress has been made in characterizing this system and confirming cell synchronization by FACSCAN analysis of DNA content. Evidence suggests that ets-1 is hyper-phosphorylated during early G2/M phase. Current cell cycle research indicates that phosphorylation/dephosphorylation of key proteins is important for progression through the cycle and entry into mitosis. The second project underway involves characterization of ets function in a human astrocytoma cell line. Preliminary findings indicate that these cells express both ets-1 and ets-2 and that carbachol, a potent activator of phosphoinositide metabolism in these cells and a mitogen for primary astrocyte cultures, leads to rapid stimulation of ets phosphorylation. This finding suggests that ets may be involved in mitogenic signalling in astrocytes, as well as lymphocytes.